Analysis of Biomarkers of DNA Damage and Mutagenicity in Mice Exposed to Acrylonitrile.

Acrylonitrile (ACN), which is a widely used industrial chemical, induces cancers in the mouse via unresolved mechanisms. For this report, complementary and previously described methods were used to assess in vivo genotoxicity and/or mutagenicity of ACN in several mouse models, including (i) female mice devoid of cytochrome P450 2E1 (CYP2E1), which yields the epoxide intermediate cyanoethylene oxide (CEO), (ii) male lacZ transgenic mice, and (iii) female (wild-type) B6C3F1 mice. Exposures of wild-type mice and CYP2E1-null mice to ACN at 0, 2.5 (wild-type mice only), 10, 20, or 60 (CYP2E1-null mice only) mg/kg body weight by gavage for 6 weeks (5 days/week) produced no elevations in the frequencies of micronucleated erythrocytes, but induced significant dose-dependent increases in DNA damage, detected by the alkaline (pH >13) Comet assay, in one target tissue (forestomach) and one nontarget tissue (liver) of wild-type mice only. ACN exposures by gavage also caused significant dose-related elevations in the frequencies of mutations in the hypoxanthine-guanine phosphoribosyltransferase (Hprt) reporter gene of T-lymphocytes from spleens of wild-type mice; however, Hprt mutant frequencies were significantly increased in CYP2E1-null mice only at a high dose of ACN (60 mg/kg) that is lethal to wild-type mice. Similarly, drinking water exposures of lacZ transgenic mice to 0, 100, 500, or 750 ppm ACN for 4 weeks caused significant dose-dependent elevations in Hprt mutant frequencies in splenic T-cells; however, these ACN exposures did not increase the frequency of lacZ transgene mutations above spontaneous background levels in several tissues from the same animals. Together, the Comet assay and Hprt mutant frequency data from these studies indicate that oxidative metabolism of ACN by CYP2E1 to CEO is central to the induction of the majority of DNA damage and mutations in ACN-exposed mice, but ACN itself also may contribute to the carcinogenic modes of action via mechanisms involving direct and/or indirect DNA reactivity.

Evaluation of Reference Genes for Quantitative PCR in Four Tissues from Rabbits with Hypercholesterolaemia

Abstract Rabbit with hypercholesterolaemia is an important model for studying cholesterol metabolism disease. This study aimed to evaluate the expression stability of nine reference genes for quantitative PCR (qPCR) analysis in adrenal gland, liver, spleen, and kidney tissue from rabbits with hypercholesterolaemia. In total, 30 male Harbin Large White (HLW) rabbits were fed a normal feed (n = 15) or a high cholesterol feed (n = 15) for 8 weeks to induce hypercholesterolaemia. Nine reference genes were verified by qPCR using cDNA extracted from rabbit tissue samples. For qPCR analysis, reference genes were evaluated using the RefFinder and GeNorm algorithms. Overall, seven rabbits with hypercholesterolaemia were identified based on body weight and total cholesterol measurements. Combining the results of the RefFinder and GeNorm algorithms, the most stable reference genes were hypoxanthine phosphoribosyltransferase 1 (Hprt1) and eukaryotic translation elongation factor 1 alpha 1 (Eef1a1) in the adrenal gland, β-2-microglobulin (B2m) and glyceraldehyde-3-phosphate dehydrogenase (Gapdh) in the liver, tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein zeta (Ywhaz) and Gapdh in the spleen, and peptidylprolyl isomerase (Ppia), β-actin (Actb), succinate dehydrogenase complex subunit A flavoprotein (Sdha), and B2m in the kidney. Taken together, our results confirmed that Hprt1 and Eef1a1, B2m and Gapdh, Ywhaz and Gapdh, and Ppia, Actb, Sdha, and B2m were the best reference genes for qPCR analyses in adrenal gland, liver, spleen, and kidney tissue, respectively, of rabbits with hypercholesterolaemia.

Expression of matrix metalloproteinases 2 and 9 in human gastric cancer and superficial gastritis.

AIM: To assess expression of matrix metalloproteinases 2 (MMP2) and MMP9 in gastric cancer, superficial gastritis and normal mucosa, and to measure metalloproteinase activity. METHODS: MMP2 and MMP9 mRNA expression was determined by quantitative real-time polymerase chain reaction. Normalization was carried out using three different factors. Proteins were analyzed by quantitative gelatin zymography (qGZ). RESULTS: 18S ribosomal RNA (18SRNA) was very highly expressed, while hypoxanthine ribosyltransferase-1 (HPRT-1) was moderately expressed. MMP2 was highly expressed, while MMP9 was not detected or lowly expressed in normal tissues, moderately or highly expressed in gastritis and highly expressed in cancer. Relative expression of 18SRNA and HPRT-1 showed no significant differences. Significant differences in MMP2 and MMP9 were found between cancer and normal tissue, but not between gastritis and normal tissue. Absolute quantification of MMP9 echoed this pattern, but differential expression of MMP2 proved conflictive. Analysis by qGZ indicated significant differences between cancer and normal tissue in MMP-2, total MMP-9, 250 and 110 kDa bands. CONCLUSION: MMP9 expression is enhanced in gastric cancer compared to normal mucosa; interpretation of differential expression of MMP2 is difficult to establish.

Reference genes for gene expression studies on non-small cell lung cancer.

STUDY OBJECTIVE: The aim of this study was to test a panel of 6 reference genes in order to identify and validate the most suitable reference genes for expression studies in paired healthy and non-small cell lung cancer tissues. METHOD: Quantitative real-time PCR followed by the NormFinder- and geNorm-based analysis was employed. The study involved 21 non-small cell lung cancer patients. RESULTS: The analysis of experimental data revealed HPRT1 as the most stable gene followed by RPLP0 and ESD. In contrast, GAPDH was found to be the least stable gene. HPRT1 together with ESD was revealed as the pair of genes introducing the least systematic error into data normalization. Validation by bootstrap random sampling technique and by normalizing exemplary gene expression data confirmed the results. CONCLUSION: Although HPRT1 and ESD may by recommended for data normalization in gene expression studies on non-small cell lung cancer, the suitability of selected reference genes must be unconditionally validated prior to each study.

Selection of reference genes for gene expression studies in human neutrophils by real-time PCR.

BACKGROUND: Reference genes, which are often referred to housekeeping genes, are frequently used to normalize mRNA levels between different samples. However the expression level of these genes may vary among tissues or cells, and may change under certain circumstances. Thus the selection of reference gene(s) is critical for gene expression studies. For this purpose, 10 commonly used housekeeping genes were investigated in isolated human neutrophils. RESULTS: Initial screening of the expression pattern demonstrated that 3 of the 10 genes were expressed at very low levels in neutrophils and were excluded from further analysis. The range of expression stability of the other 7 genes was (from most stable to least stable): GNB2L1 (Guanine nucleotide binding protein, beta polypeptide 2-like 1), HPRT1 (Hypoxanthine phosphoribosyl transferase 1), RPL32 (ribosomal protein L32), ACTB (beta-actin), B2M (beta-2-microglobulin), GAPD (glyceraldehyde-3-phosphate dehydrogenase) and TBP (TATA-binding protein). Relative expression levels of the genes (from high to low) were: B2M, ACTB, GAPD, RPL32, GNB2L1, TBP, and HPRT1. CONCLUSION: Our data suggest that GNB2L1, HPRT1, RPL32, ACTB, and B2M may be suitable reference genes in gene expression studies of neutrophils.

Transcriptional responses of murine macrophages to infection with Yersinia enterocolitica.

Transcriptional responses of J774 murine macrophage-like cells to infection with Yersinia enterocolitica were evaluated with oligonucleotide microarrays interrogating 12 488 genes and expressed sequence tags. Virulence plasmid (pYV)-cured yersiniae induce a transcriptional programme resembling a general inflammatory response. pYV-carrying yersiniae translocating the Yersinia outer proteins (Yops) impact on this transcriptional programme in two ways: first, by suppressing this inflammatory response and, secondly, by inducing sustained expression of a distinct set of genes with known silencing functions. These tranquilizing patterns of gene expression could be confirmed by real-time reverse transcription polymerase chain reaction, are stable upon reduction in bacterial load and could also be reproduced in BALB/c-derived bone marrow macrophages. Prestimulation of macrophages with interferon (IFN)-gamma, but not with interleukin (IL)-4, induces partial resistance against pYV-mediated transcriptional tranquilization. The first effect, suppression of the inflammatory programme, is mediated by YopP, whereas no YopH- or YopM-regulated genes could be identified under our stringent statistical criteria. The bacterial protein responsible for the second effect, induction of silencing genes, remains elusive. We suggest that Yersinia enterocolitica might use two independent mechanisms to inhibit macrophage inflammatory responses at the transcriptional level.

The role of various biomarkers in the evaluation of styrene genotoxicity.

We evaluated our data on the occupational exposure to styrene in lamination workers. The battery of parameters included markers of external and internal exposure and biomarkers of biological effects and susceptibility. DNA repair capacities have been determined in both exposed and control groups. Styrene workplace concentration significantly correlated with styrene concentration in blood, exhaled air and urinary mandelic acid. Haemoglobin and O(6)-styrene oxide (SO)-guanine DNA adducts were significantly higher in exposed subjects as compared to controls and correlated with exposure parameters. In styrene-exposed workers 1-SO-adenine DNA adducts were detected (2.6 per 10(9) dNp), while in controls these adducts were below the detection limit. 1-SO-adenine adduct levels were affected by both acute and cumulative exposure (P=0.001, F=86.0 and P=0.017, F=59.0, respectively) and associated with cytochrome P450 2E1 (CYP2E1) polymorphisms (R(2)=0.442). Mutant frequencies (MF) at the hypoxanthine-guanine phosphoribosyltransferase (HPRT) locus appeared to accumulate with exposure over time and were associated with glutathione S-transferase P1 (GSTP1) polymorphism. DNA repair capacity increased with the exposure, except for the group exposed to the highest styrene concentration. In this particular group, increased DNA repair capacity to remove oxidative DNA damage was found.

Cell imaging and morphology: application to studies of inherited purine metabolic disorders.

A number of inherited or drug-induced metabolic disorders involving dysfunctions in purines and pyrimidines are strongly associated with neurological dysfunction, e.g., Lesch Nyhan syndrome. Such disorders have been studied extensively using biochemical and molecular techniques in order to examine how such defects occur, sometimes using in vitro models based upon cultured neuroblastoma cell lines. However, these metabolic dysfunctions may manifest their effects in other ways, such as impaired synaptic transmission and gross abnormalities in neuronal growth and differentiation. This review outlines the latter novel facet of purine research. It is proposed that by employing cell imaging techniques and cultured neuroblastoma cell lines, believed to model the nervous system, significant insights into how inherited disorders of purine metabolism affect neuronal development can be obtained. This review provides an example of the application of these techniques to understand the etiology of Lesch Nyhan syndrome, and encourages further study of the role of purines and pyrimidines in the development of the nervous system.

Ion-exchange column chromatographic method for assaying purine metabolic pathway enzymes.

High energy phosphate levels fall rapidly during cardiac ischemia and recover slowly (more than one week) during reperfusion. The slow recovery of ATP may reflect a lack of purine metabolic precursors and/or increased activity of purine catabolic enzymes such as 5'-nucleotidase (5'-NT, EC 3.1.3.5) and adenosine deaminase (ADA, EC 3.5.4.4). The activity of enzymes involved in both the catabolism of ATP precursors (5-NT and ADA) and the restoration of ATP from slow synthetic pathways [adenosine kinase (AK, EC 2.7.1.20), adenine phosphoribosyl transferase (APRT, EC 2.4.2.7) and hypoxanthine phosphoribosyl transferase (HPRT, EC 2.4.2.8)] may directly affect the rate of ATP recovery. Strategies to enhance recovery will depend on the relative activity of these enzymes following ischemia. Their activity in different species and their response to ischemia are not well characterized. Hence, rapid assay methods for these enzymes would facilitate detailed time course studies of their activities in postischemic myocardium. We modified a single ion-exchange column chromatographic method using DEAE-Sephadex to determine the products of incubation of 5'-NT, AK, APRT and HPRT with their respective substrates. The uniformity of the final product measurement procedure for all assays permits the activities of the four enzymes to be rapidly determined in a single tissue sample and facilitates the study of a large number of samples. This technique should also be useful for enzymes of the pyrimidine metabolic pathway.

Mice deficient for 5-lipoxygenase, but not leukocyte-type 12-lipoxygenase, display altered immune responses during infection with Schistosoma mansoni.

Periovular granuloma formation during Schistosoma mansoni infection is a complex, multifaceted immunologic response. Products of arachidonic acid metabolism have been shown to contribute to this response through studies in which general inhibitors of lipoxygenase function reduce granulomatous inflammation. To determine which lipoxygenases are important for granuloma development in schistosomiasis, wild type mice or mice deficient for 5-lipoxygenase (5-LO) or "leukocyte-type" 12-lipoxygenase (12-LO) were infected with S. mansoni and studied for responses to schistosome eggs and egg antigens. At the acute stage of infection, when granuloma formation is usually maximal, 5-LO deficient mice developed smaller granulomas around liver-deposited schistosome eggs compared with wild type or 12-LO deficient mice. 5-LO mice also displayed less antibody-mediated (5 h) and cell-mediated, delayed-type (24 h) hypersensitivity to schistosome egg antigens than did the other two infection groups. In an attempt to determine possible mechanisms for the reduced inflammatory responses, we also measured hepatic mRNA levels of cytokines that have been shown to influence granuloma size (IL-4, IL-10, and IFN-gamma). The mRNA levels for IL-10 were significantly lower in 5-LO-deficient mice, but SEA-stimulated spleen cells did not demonstrate a significant difference in IL-10 production between wild type and 5-LO mice. These data suggest that 5-LO plays a role in host responses to schistosomiasis via a mechanism that cannot be explained solely by changes in expression of these cytokines.

Ectopically expressed CAG repeats cause intranuclear inclusions and a progressive late onset neurological phenotype in the mouse.

The mutations responsible for several human neurodegenerative disorders are expansions of translated CAG repeats beyond a normal size range. To address the role of repeat context, we have introduced a 146-unit CAG repeat into the mouse hypoxanthine phosphoribosyltransferase gene (Hprt). Mutant mice express a form of the HPRT protein that contains a long polyglutamine repeat. These mice develop a phenotype similar to the human translated CAG repeat disorders. Repeat containing mice show a late onset neurological phenotype that progresses to premature death. Neuronal intranuclear inclusions are present in affected mice. Our results show that CAG repeats do not need to be located within one of the classic repeat disorder genes to have a neurotoxic effect.

Purine nucleotide metabolism: specific aspects in chronic lymphocytic leukemia lymphocytes.

The metabolism of purine nucleotides was studied in human peripheral blood lymphocytes from healthy subjects and patients with B-cell chronic lymphocytic leukemia. Nucleotide content was determined by HPLC. The rate of de novo synthesis of purine nucleotides was measured kinetically by following the incorporation of 14C-formate into the nucleotides of a lymphocyte suspension. The patterns of the main enzymes involved in purine nucleotide metabolism (those of the salvage pathway and catabolism) were estimated by a radiochemical method. Although the data expressed in relation to cells and protein showed some discrepancies, several common differences were evident in both cases. The main differences were an increase in NAD and IMP, a sharp decrease in 5'-nucleotidase activities and in total guanylate content and synthesis, and an increase in the A/G ratio in lymphocytes of patients with respect to controls. The changes in these parameters in CLL indicate an imbalance in purine metabolism and may play a specific role in the biology of the leukemia cell. They are also potential biochemical markers of lymphoid malignancies and may be useful in chemotherapic applications.

Nitrate contamination of drinking water: relationship with HPRT variant frequency in lymphocyte DNA and urinary excretion of N-nitrosamines.

We studied peripheral lymphocyte HPRT variant frequency and endogenous nitrosation in human populations exposed to various nitrate levels in their drinking water. Four test populations of women volunteers were compared. Low and medium tap water nitrate exposure groups (14 and 21 subjects) were using public water supplies with nitrate levels of 0.02 and 17.5 mg/l, respectively. Medium and high well water nitrate exposure groups (6 and 9 subjects) were using private water wells with mean nitrate levels of 25 and 135 mg/l, respectively. Higher nitrate intake by drinking water consumption resulted in a dose-dependent increase in 24-hr urinary nitrate excretion and in increased salivary nitrate and nitrite levels. The mean log variant frequency of peripheral lymphocytes was significantly higher in the medium well water exposure group than in the low and medium tap water exposure groups. An inverse correlation between peripheral lymphocyte labeling index and nitrate concentration of drinking water was observed. Analysis of N-nitrosamine in the urine of 22 subjects by gas chromatography-mass spectrometry revealed the presence of N-nitrosopyrrolidine in 18 subjects. Analysis of the mutagenicity of well water samples showed that a small number of the well water samples were mutagenic in the Ames Salmonella typhimurium test after concentration over XAD-2 resin. In conclusion, consumption of drinking water, especially well water, with high nitrate levels can imply a genotoxic risk for humans as indicated by increased HPRT variant frequencies and by endogenous formation of carcinogenic N-nitroso compounds from nitrate-derived nitrite.

The majority of T lymphocytes are polyclonal during the chronic phase of chronic myelogenous leukemia.

To clarify the extent of cell lineage involvement in chronic myelogenous leukemia (CML), we investigated the bcr gene rearrangement and clonality using the X-chromosome-linked restriction fragment length polymorphism (RFLP) methylation method in T lymphocytes and granulocytes. We examined the granulocyte and T-cell fractions from the peripheral blood of seven female patients with CML during the chronic phase; patients were heterozygous for RFLPs at the phosphoglycerate kinase (PGK) or the hypoxanthine phosphoribosyltransferase (HPRT) gene. RFLP-methylation analysis of granulocytes demonstrated a monoclonal pattern in six of the seven patients and a rearranged bcr gene in all seven patients. In contrast, T lymphocytes exhibited a polyclonal pattern in six cases; in one case, a faint band was observed following methyl-sensitive enzyme cleavage. The bcr gene analysis in T lymphocytes showed the germline in every case. Our results indicate that the majority of T lymphocytes are polyclonal during the chronic phase of CML and confirm previous reports based on glucose-6-phosphate dehydrogenase, cytogenetic, and bcr rearrangement analyses.

Mutational specificity and cancer chemoprevention.

Mutational specificity describes the composite of all of the genetic alterations in a collection of mutations arising from a specific treatment. The information includes not only the nature of the genetic change (e.g., a base substitution or a frameshift), but also information about nucleotide position and hence the DNA context. As both the type of DNA damage and its position can be expected to reflect the nature of the chemical and physical mutagen, mutational specificity can be expected to provide insights into mechanisms of mutation. Conversely, mutational spectra should also provide insights into the identity of the mutagen. Indeed, the pioneering work on mutational specificity in Escherichia coli indicates that each physical or chemical treatment produces a unique spectrum of mutations. With the application of biotechnology to the field of genotoxicology, the database of sequenced mutations has become quite substantial. Both in vitro and in vivo data has been obtained following exposure to a variety of agents. In this communication we will critically assess whether the reality of mutational specificity has fulfilled the expectations and to examine what potential remains to be explored, especially in the area of monitoring human populations. The usefulness of both mutational spectra analysis and population monitoring with regards to chemoprevention are discussed.

Enzymes of purine salvage and catabolism in the mouse preimplantation embryo measured by high performance liquid chromatography.

The activities of five enzymes involved in purine salvage and catabolism--hypoxanthine phosphoribosyl transferase (HPRT), adenine phosphoribosyl transferase (APRT), adenosine deaminase (ADA), purine nucleoside phosphorylase (PNP) and guanase--were measured in mouse embryo extracts, from the one-cell to the blastocyst stage. Xanthine oxidase activity was not detected. The analyses were performed using high performance liquid chromatography and the enzymes showed different patterns of activity during development. Activities of HPRT, APRT and PNP were low before morula formation, and then increased until the blastocyst stage. ADA and guanase showed high activities after fertilization; guanase activity decreased sharply after the two-cell stage and ADA activity decreased sharply after the morula stage. Blastocyst formation was accompanied by a further decline in activity of both enzymes. The methods used may be suitable for measuring these enzymes in single human embryos, or in biopsies derived from them.

Molecular analysis of clonality in plasma cell dyscrasias.

It has been suggested that multiple myeloma, generally considered a neoplastic disorder of mature plasma cells, may arise from a pluripotent haemopoietic stem cell. The possibility that circulating lymphocytes derive from the same neoplastic progenitor has been tested in a large number of studies in the past few years, as proof of the interest that this subject is raising among scientists, and also of its elusiveness. We studied a group of 29 patients with plasma cell dyscrasias in order to evaluate clonality of haemopoietic cell populations. The X-linked markers hypoxantine phosphoribosyltransferase (HPRT) and phosphoglycerate kinase (PGK) disclosed no monoclonal component in seven heterozygous women. Analysis of immunoglobulin gene rearrangement with four probes showed a germline configuration in samples from 25/29 patients. Only four bone marrow samples from subjects with aggressive disease had rearranged C mu sequence; one had rearrangement of JH and C mu.

Purine metabolism and B-lymphocyte development in the chicken bursa of fabricius.

The activity of three enzymes involved in the salvage pathway of purine nucleosides--purine nucleoside phosphorylase (PNP), xanthine dehydrogenase (XDH), and hypoxanthine-guanine phosphoribosyl transferase (HGPRT)--was investigated in cellular fractions of the chicken bursa of Fabricius differentially enriched in epithelial cells or lymphocytes. Markedly increasing levels of PNP and XDH were observed along with the enrichment in epithelial cells together with a slight, though significant, decrease in HGPRT activity. By contrast, a dramatic fall in PNP and XDH activities was detected along with the enrichment in lymphocytes together with a slight, though significant, increase in HGPRT activity. This sharply different distribution of the three enzymes, all sharing hypoxanthine as a substrate, clearly indicates that lymphocytes preferentially channel hypoxanthine into the salvage and interconversion pathways, phosphorylating it to IMP, while epithelial cells rapidly catabolize such a purine base to uric acid. Moreover, epithelial cells, unlike lymphocytes, are able to retain high intracellular levels of both hypoxanthine and inosine. These results support the possibility that epithelial cells contribute to the normal development of bursal lymphocytes by supplying such actively proliferating cells with purine rings and at the same time by preventing them from accumulating potentially toxic high levels of purine nucleotides being able to rapidly eliminate excess hypoxanthine as uric acid from the bursa environment into the bloodstream.

Changes in permissiveness for the expression of microinjected DNA during the first cleavages of mouse embryos.

LacZ DNA and LacZ RNA were microinjected during the first cleavages of embryos. LacZ DNA was not expressed before 18-19 h post insemination (hpi) but LacZ RNA was translated. Before 22 hpi LacZ DNA was expressed in the pronuclei of the one-cell embryos and the polypeptides of the minor, but not the major activation period of the genome were synthesized. This suggests a negative control of transcription before 18-19 hpi and demonstrates that its resumption is independent of the first cleavage and of the major activation of the genome. At the time of the minor activation the eggs contain the trans-acting elements to express a variety of genes that they do not express. It may indicate that, the minor and the major activation of the genome are differently controlled.